In the past decades, the development of an efficient vaccine for the prevention of viral infectious diseases has been one of the main objects of medicine. In the preparation of live virus vaccines virus mutants are used which have antigens identical to that of the wild type virus, yet have a reduced pathogenicity or virulence.
To prepare dead vaccines, wild type viruses are inactivated by a physical or chemical treatment, such as with formalin, hydroxylamine, .beta.-propion lactone or UV radiation, the type of inactivation being important, in particular with a view to retaining the immunogenicity of the viral antigens. The technique mostly used for virus inactivation in the preparation of vaccines is the mild treatment of viruses with formalin, which, however, requires a long incubation period of up to 15 days (e.g. in case of HAV) so as to obtain a sufficient reduction of virus activity.
Whole virus vaccines generally stimulate the development of circulating antibodies to the enveloping proteins of the virus. Thus it has repeatedly been pointed out that it must be ensured in this inactivation process that the immunogenicity of the viral proteins is retained. Simultaneously, however, it must be ensured that all the viruses in the preparation are inactivated so as to guarantee safe vaccines.
As a consequence of incidents caused by incompletely inactivated whole virus vaccines or contaminations of the vaccine preparation with cellular components or undesired viral particles, there has been a search for alternative inactivation methods (Horaud, Develop. Biol. Standard. 75 (1991), 3-7; Brown, Develop. Biol. Standard. 81 (1993), 103-107).
Since formalin inactivation is not always complete, and since residual infectious viruses have been found in individual vaccine preparations (Smith et al., Am. J. Hyg. 63 (1956), 150-164), Mussgay et al. (Inter-virology 1 (1973), 259-268) have proposed a two-step inactivation procedure, wherein a formalin inactivation followed upon a treatment with Tween 80/ether, NP 40 or deoxycholate. However, they found that by the additional treatment with the detergent or solvent/detergent, the protectivity of the formalin-inactivated vaccine was negatively affected.
Thus, in recent years, subunit vaccines have increasingly been developed alternatively to vaccines containing inactivated whole viruses. In the preparation of subunit vaccines, the intact virus is solubilized with a strong detergent, the viral proteins are dissolved out of the virus, and selective antigens which are capable of stimulating protective antibodies are isolated and used for vaccine preparation. Simultaneously, non-viral proteins and membrane components of the virus that might have undesired side effects when administering the vaccines are eliminated. The antigens isolated for the subunit vaccines may also be contained in the vaccine in highly purified form and at a high concentration. Yet the immunogenicity may be reduced as compared to a whole virus vaccine, since only individual antigens are available as an immunogen.
To induce a sufficient immune response at vaccination, so-called split vaccines have thus been prepared. There, the virus is completely solubilized with a detergent or with a mixture of a detergent and a solvent, the integrity of the virus is destroyed, and the virus is dissolved into its individual components, such as core proteins, enveloping proteins and membrane components. The thus prepared mixture of viral components is then utilized for vaccination.
Experiments carried out in the course of virus inactivation of human plasma products have shown that a solvent/detergent treatment with TNBP/Tween.RTM. 80 is capable of inactivating lipid-enveloped viruses, such as, e.g., HIV, HCV, HBV or Sindbis virus (Piet et al., Transfusion 30 (1990), 591-598).
In the commercial production of influenza virus subunit vaccines, various methods are used for the inactivation with lipid solvents, such as Triton.RTM. X-100, cetyltrimethyl-ammonium bromide, TNBP/Tween.RTM. 80 or diethyl ether/TweenO 80. Particularly when using diethyl ether in the inactivation step, it has been found that there resulted a marked decrease of the hemagglutinin activity. Danihelkova et al. (Acta virol. 28 (1984), 26-32) found that the hemagglutinin and neuraminidase activities of inactivated influenza virus are retained after a treatment with TNBP/Tween.RTM. 80 or diethyl ether/Tween.RTM. under certain conditions. They note, however, that removal of the solvent, in particular of diethyl ether, from the preparation poses problems, and therefore an inactivation method using TNBP/Tween.RTM. 80 is considered to be preferable.
To avoid the complex removal of the solvent, thus only Triton X-100 was used as non-ionic detergent in the preparation of an influenza split vaccine (Gross et al., J. Clin. Microbiol. 14 (1981), 534-538).
Non-ionic detergents do not have charged groups, the hydrophilic character of these detergents is caused by the hydroxyl group. In contrast to ionic detergents, they solubilize membrane proteins much more mildly. The non-ionic detergent breaks up lipid-lipid and lipid-protein bonds, while protein-protein interactions remain unaffected. By this, the native structure of the proteins is retained. Moreover, the detergent replaces the lipids which normally are connected to the hydrophobic portion of the proteins, whereby a lipid-like environment is created and thus the solubilized proteins are stabilized.
Investigations regarding the protectivity of inactivated SIV vaccines after a treatment with formalin, psoralen, Triton.RTM. X-100 or Tween.RTM./ether indicated that formalin and psoralen-inactivated SIV preparations do not protect against an SIV infection and that the Triton.RTM. X-100 inactivation yields only partial protection. However, with a Tween.RTM. 80/ether inactivated split vaccine of SIV, a protective effect could be shown only at a high antigen dose and in the presence of a potent adjuvant (Stahl-Hennig et al., Virology 186 (1992), 588-596).
Particularly when using infectious human pathogenic viruses for preparing a whole virus vaccine it is necessary to provide an effective and safe method of inactivating the viruses. This particularly holds for such viruses as, e.g., HIV, in which an inactivation with conventional methods has been considered to be insufficient. As mentioned above, in case of SIV, e.g., a formalin inactivation leads to a possible loss of antigenicity.